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標題: | 探討栽培種草莓在水楊酸誘導下所引發之抗病防禦網絡 Uncovering salicylic acid-mediated defense network in cultivated strawberry |
作者: | Jui-Yu Liao 廖睿瑜 |
指導教授: | 鍾嘉綾(Chia-Lin Chung) |
關鍵字: | Fragaria x ananasa,水楊酸,抗性網絡,轉錄體定序, Fragaria x ananasa,salicylic acid,defense network,transcriptome sequencing, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 草莓為高經濟及高營養價值之水果,因其基因體大小相對簡易且可快速繁殖,適
合做為薔薇科果樹研究的模式植物。適逢近年全球暖化,臺灣草莓育苗期亦因病 害嚴重而損失慘重 (如:炭疽病等),當前並無可完全抵禦之品系。為克服此一困 難,開發新穎性防治策略因應,須對草莓 (尤其本土栽培品種) 之抗感病機制有所 瞭解。我們選定對多種病害中度感病的臺灣本土栽培種「桃園三號」與日本強健 抗病品種「Superjumbo」為材料。為能完整解析草莓的防禦系統,本研究運用次世代定序技術,針對水楊酸在兩種草莓品系上所誘導之基因表現,以高通量方式進 行mRNA-seq whole-transcriptome analysis,以建構抗性相關基因資料庫。為確保定序樣品之防禦反應確實已被啟動,我們首先測定以水楊酸處理兩品種草莓之反應,結果證實可造成植株對炭疽病抗性增強及抗性途徑下游基因 (包含FaPR1、FaOLP2、和FaWRKY1) 之表現量增加。選定處理後6 hr、12 hr、24 hr 和48 hr 等時間點之RNA 樣品加以混合再進行定序,以獲取全面性資訊。考量栽培種草莓基因體,屬複雜之八倍體,因此本研究在基因組裝策略上,分別對de novo assembly 和gene annotation 進行策略評估。de novo assembly 部分,運用Trinity、Velvet/Oases 及CLC Bio 三種軟體,配合multiple k-mer 測試,結果指出Trinity 較可適用於八倍體草莓﹔gene annotation 部分,則分別依據二倍體草莓基因體資料庫及NCBI nr database 進行,結果顯示以二倍體草莓基因體資料庫進行註解可獲得較完整之資訊。依據Gene Ontology 及註解結果,我們總共於兩品種中篩選出近800 筆處理水楊酸後具差異性表現之基因,包括可能參與在水楊酸生合成、植物與病原交互作用、和防禦訊息傳導等重要功能之基因,以及數個具高度差異表現之新穎性未註解基因。透過quantitative real-time reverse transcription PCR (qRT-PCR),目前已驗證了資料庫中的17 個分屬於水楊酸抗病網路不同途徑之重要基因,包含genes encoding Resistance (R) protein、Shikimate dehydrogenase、Shikimate kinase、Chorismate mutase、Flavanone 3-dioxygenase、NPR1、FaWRKY1、WRKY70、WRKY51、TGA6、GrxC9、FaOLP2、FaPR1、Brassinosteroid insensitive 1-associated receptor kinase 1、Interleukin-7 receptor、Hyoscyamine 6-dioxygenase 和Patatin T5 等,確實可受到水楊酸誘導而引發表現量上升,並在兩品種間呈現表現時間之差 異性。此等基因透過次世代定序及qRT-PCR 測得之表現趨勢,與少數過去文獻相互比較印證後,可說明本研究所建構抗性資料庫之可信度。本研究為首度全面性深入探討栽培種草莓水楊酸誘導抗性網絡之研究,相關結果將有助於了解草莓受不同病原菌入侵時可能啟動之水楊酸抗性途徑,以利未來於抗病育種及防治策略開發等方面之運用。 Cultivated strawberry, an economically important small fruit, has been considered a potential model system for Rosaceae plants. Pest damage and pesticide residue problems have always been major threats to strawberry cultivation in Taiwan. Aiming to develop novel disease control strategies, we used the Illumina next generation sequencing technology for high-throughput identification of genes up- and down-regulated by defense hormone- salicylic acid (SA), in two strawberry cultivars, Taoyuan 3 and Superjumbo. We verified the effectiveness of SA-treatment by evaluating anthracnose resistance and the expression of FaPR1, FaWRKY1, and FaOLP2, the hallmark genes downstream of the SA signaling pathway, in SA- or ddH2O-pretreated plants. After that, total RNA was extracted from samples of each cultivar collected 6, 12, 24, and 48 hours after SA- or ddH2O-treatment and then pooled for sequencing. To tackle the complex octoploid genome, we adopted an assemble-then-align strategy. We first assessed Trinity, Velvet/Oases and CLC Bio in combination with multiple k-mer strategy for their efficiencies in de novo assembly. Trinity generated more full-length transcripts across a broad range of expression levels, with the assemblies exhibiting a higher similarity to the corresponding genes in a reference diploid strawberry genome. The result indicated that Trinity may be a better assembler for dealing with the homologous/homoeologous genes in the octoploid strawberry genome. As for gene annotation, we found that more transcripts could be better-annotated with the use of the diploid strawberry genome database. Overall, we identified about 800 genes that were significantly differentially expressed between SA-treated plants and the control. These included several novel genes and numerous genes likely involved in SA biosynthesis, plant-pathogen interactions, and defense signaling. We investigated the time-course expression profiling of a set of selected candidate genes using real-time quantitative RT-PCR. The expression of seventeen defense-related genes, encoding Resistance (R) protein, Shikimate dehydrogenase, Shikimate kinase, Chorismate mutase, Flavanone 3-dioxygenase, NPR, FaWRKY1, WRKY70, WRKY51, TGA6, GrxC9, FaOLP2, FaPR1, Brassinosteroid insensitive 1-associated receptor kinase 1, Interleukin-7 receptor, Hyoscyamine 6-dioxygenase, and Patatin T5, has been verified to be highly induced in response to SA. The expression patterns of these genes, quantified by RNAseq and qRT-PCR, were consistent with those previously reported in model plants and/or strawberry, suggesting that the defense database we constructed should be valid. This study is the first comprehensive investigation of the SA-mediated pathway in cultivated strawberry. The results will contribute to a better understanding of strawberry resistance mechanisms against the invasion of biotrophic/hemibiotropic pathogens. The identified defense-related genes can also be used as selection markers in strawberry resistance breeding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78231 |
全文授權: | 有償授權 |
電子全文公開日期: | 2023-08-08 |
顯示於系所單位: | 植物病理與微生物學系 |
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